The interaction of heme-containing proteins and nitric oxide and regulatory functions of the latter in changing the activity of enzymes while interacting with them, as well as the biological role of nitrosyl complexes of hemoproteins as means of depositing nitric oxide are described, for example, in the article [1].
Dinitrosyl iron complexes (DNIC) are considered to be a relatively stable form of NO in cells. Thiol groups of proteins or low-molecular-weight thiols (e.g., cysteine or glutathione) are involved in the formation of these complexes [2]. Such complexes are formed in macrophages and endothelial cells. They are considered as the main depot (pool) of NO in the body [3]. Experimental evidence suggest that NO can be released from endothelial cells in the form of dinitrosyl iron complexes (DNIC) rather than in an arbitrary form. Low-molecular-weight mercaptans (e.g., cysteine or glutathione) are known to compete with thiol groups of proteins for the formation of nitrosyl complexes. Finally, the profile of properties of dinitrosyl iron-sulfur complexes is characterized by the fact that they have practically the same physiological activity and stability as endothelium-derived relaxing factor (EDRF) and, therefore, can be involved in the biological action of NO [4]. NO is able to alter the activity of enzymes by interacting with their functionally important groups and, primarily, with heme iron (Fe-heme) and thiols. The activation of guanylate cyclase is the most striking example of this. Binding of NO to heme iron of the regulatory subunit of guanylate-cyclase causes a disruption in the bond between iron and nitrogen of a histidine imidazole group resulting in changes in both the structure of the active center and protein conformation [5]. The activity of enzyme increases tenfold leading to an increase in the level of cyclic guanosine monophosphate (cGMP). If platelets are target cells, then increasing cGMP causes a reduction in blood coagulation. In case of a smooth muscle cell, an increase in intracellular cGMP leads to a relaxation of smooth muscle. This mechanism underlies physiological phenomena, such as regulation of vascular tone, regulation of Oddi's sphincter tone in the duodenum and some other phenomena. As a regulator of vascular tone (and, in such way, of blood pressure), NO is involved in the pathogenesis of various cardiovascular diseases, including hypertension and atherosclerosis. NO is well known by its protective role at the initial stage of ischemia as a factor improving blood circulation and reducing tissue damage [6]. The ability of NO to affect the pulmonary blood flow and bronchial tone has been found to be therapeutic [7]. For example, S-nitrosoglutamate (a natural NO metabolite) regulates the air resistance of the bronchi [8]. As a neurotransmitter of the peripheral nervous system, NO provides reproductive functions in men and can play a crucial role in the treatment of impotence [9]. NO is involved in inflammatory and immune processes. Thus, macrophages activated by γ-interferon, tumor necrosis factors (TNF) and lipopolysaccharides (LPS) dramatically increase the synthesis of NO and ONOO—, damage bacterial cells and, in this way, provide antimicrobial action. At the same time, in case of sepsis, formation of NO in toxic quantities plays a negative role. Reduced vascular tone and inevitable fall in blood pressure under the action of NO can become critical and lead to shock. In case of ischemia/reoxygenation, increased synthesis of NO and ONOO—causes tissue damage and cell death [10]. NO toxicity at the cellular level is associated with the formation of nitrosyl complexes of heme proteins and/or their S-nitrosylation. The inhibition of enzymes of the respiratory chain, Krebs cycle, and DNA synthesis is a consequence of such modifications. Further, the development of oxidative stress, in the presence of NO, is associated with the production of a powerful oxidant—ONOO—which irreversibly suppresses enzymes and oxidizes lipids and DNA. Thus, on the one hand, NO can act as a pro-oxidant due to the formation of ONOO—. On the other hand, NO is an interceptor of free radicals and a reducing agent and, as such, may play the role of an antioxidant. NO readily reacts with other free radicals and thus can cause either an interruption in the lipid peroxidation chain or an inhibition of its initiation [11, 12]. In a number of pathophysiological processes occurring with the involvement of NO, nitrosyl complexes of heme-containing proteins can play an important role. This primarily refers to conditions associated with circulatory disorders—ischemia, hypertension, or shock—which are associated with the formation of nitrosyl complexes—guanylate-cyclase. A reduction in cellular respiration and increased production of free radicals by mitochondria in inflammatory and neurodegenerative processes may be associated with the formation of nitrosyl complexes of cytochromes of the electron transport chain [13].
The following analogues used for obtaining a pharmacologically active complex with nitric oxide are known to the applicant.
The prior art describes a composition for the release of nitric oxide (NO) which, if necessary, allows for the quick release of nitric oxide and, at the same time, the formation of S-nitrosothiol compounds [14]. The compound provides a slower release of NO and a longer duration of action. The composition comprises a liquid phase, containing a solvent and at least one reducing agent, and a solid phase, containing nitrate and/or nitrate, copper ions, and at least one thiol. Pantetheine, alpha lipoic acid, phosphapantetheine, cysteine, homocysteine, thioglycolic acid, β-mercaptopropionic acid, β-mercaptoethanol, β-thioethanolamine, coenzyme A, cysteamide, γ-glutamylcysteine, phytogelatin, trypanothione, captopril, glutathione, and N-acetylcysteine are used as thiols suitable for the use in the said composition. The said composition can preferably be used in all clinical situations where the release of NO may affect the etiology and pathogenesis of a disorder. Particularly preferred is its use in the treatment of male sexual dysfunction, in particular erectile dysfunction.
The composition is produced by using a mixture formed by dissolving fumaric acid and ascorbic acid in propylene glycol and adding a solid phase, comprising a mixture of thiol (e.g., alpha-lipoic acid), sodium nitrite and powdered copper sulfate, to the solution followed by mixing at a room temperature. The resulting mixed solution contains propylene glycol, ascorbic acid, alpha-lipoic acid, fumaric acid, sodium nitrite, and copper sulfate residues. To use the described composition in this case, a special container is required to ensure the long-term separate storage of the liquid and the solid phase of the composition.
The release of nitric oxide (NO) and the use of thiol as a reducing agent is a common feature of the analogue and the present invention. However, the composition according to the analogue does not imply the use of hemocomplexes of liposomal cytochrome c which, in terms of its phospholipid composition, is biologically close to membrane cells of the mammalian body. A further drawback of the analogue is the use of two different phases of the composition and a special container for their storage.
The prior art discloses the use of phospholipids to produce a liposomal agent with nitric oxide (NO), where cytochrome c is used as a catalyst of nitrosylation reaction, i.e. conversion, where thiol (glutathione) is used as a substrate for nitrosylation [15]. The purpose of obtaining such a liposomal agent is to study catalytic properties of cytochrome c with nitric oxide. In the said reaction, activation of NO is achieved with a known prodrug of nitric oxide—V-PYRRO/NO, not with gas. According to [15], such agent is used only as a model close to the cell structure for purposes of research and not as a pharmacologically active agent. Nitrosothiols are also used as carriers and there is no use of cytochrome c and liposomes for this.